Forum in immunology

Immunomodulation after allogeneic bone marrow transplantation byCD4+CD25+ regulatory T cells

Petra Hoffmann a, Tina J. Boeld b, Biserka Piseshka b, Matthias Edinger b,*a Institute of Immunology, University Hospital Regensburg, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany

b Department of Hematology and Oncology, University Hospital Regensburg, University of Regensburg, Franz-Josef-Strauss-Allee 11,93053 Regensburg, Germany

Available online 31 May 2005

Abstract

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation (BMT) caused by donor T cells. Immuno-suppression mediated by CD4+CD25+ regulatory T cells has been shown to ameliorate such pathogenic immune responses in animal models.Here, we summarize recent findings from experimental and clinical studies and propose a model for peripheral tolerance induction after BMT.© 2005 Elsevier SAS. All rights reserved.

Keywords: Tolerance; Graft-versus-host disease; Leukemia

1. Introduction

Allogeneic bone marrow or stem cell transplantation (SCT)is a well-established treatment modality for malignant andnon-malignant hematologic diseases. Advances in support-ive care as well as the introduction of growth factors and newanti-infectious reagents significantly improved the safety ofthis treatment modality and reduced its early morbidity andmortality. The development of reduced-intensity and non-myeloablative conditioning regimens further diminished therisk of toxic complications associated with high-dose radio-and chemotherapy and enabled extension of the use of allo-geneic SCT to elderly patients who were previously excludedfrom this potentially life-saving therapy. Such regimens inparticular rely on immune cell-mediated mechanisms for sup-port of stem cell engraftment and early reconstitution, thustransforming allogeneic SCT from an organ-replacement strat-egy into a highly efficient cellular immunotherapy. Maturedonor T cells within the stem cell graft are the main media-tors of these beneficial immune effects, but they are alsoresponsible for the induction of graft-versus-host disease

(GVHD), a multi-organ syndrome marked by severe host tis-sue destruction, which is still a major complication after allo-geneic SCT. Up until now, standard GVHD prophylaxis andtherapy have comprised drugs that cause generalized immu-nosuppression and place patients in danger of opportunisticinfections and tumor relapse. Recent preclinical studies haveaimed at engaging the regulatory and suppressive mecha-nisms intrinsic to the donor immune system to ameliorate allo-responses without paralyzing all immune functions. A cellu-lar population with suppressive activity that has attractedconsiderable attention are CD4+ T cells which constitutivelyexpress the IL-2 receptor a-chain (CD25). Their suppressivecapacity has been demonstrated in various disease models, asdetailed in other contributions to this issue of Microbes andInfection, but also in tolerance induction after allogeneic organand hematopoietic SCT. In this review we summarize recentfindings from murine bone marrow transplantation (BMT)models concerning the role of donor-derived CD4+CD25+

regulatory T cells (Treg) for the protection from GVHD andadapt a model of peripheral tolerance induction by suppres-sor T cells that was previously deduced from studies in solidorgan transplantation. Finally, a brief summary of current dataregarding human Treg cells in allogeneic SCT is presented.

* Corresponding author. Tel.: +49 941 944 5549; fax: +49 941 944 5502.E-mail address: matthias.edinger@klinik.uni-regensburg.de

(M. Edinger).

Microbes and Infection 7 (2005) 1066–1072

www.elsevier.com/locate/micinf

1286-4579/$ - see front matter © 2005 Elsevier SAS. All rights reserved.doi:10.1016/j.micinf.2005.03.035

2. The ambiguous role of donor T cells in allogeneicSCT

The success of allogeneic SCT relies in part on immunecell-dependent mechanisms that are mainly mediated bymature donor T cells contained in the stem cell graft. Suchdonor T cells facilitate the engraftment of donor stem cellsby eliminating residual host lympho- and myelopoiesis,thereby also promoting potent graft-versus-tumor effects inpatients transplanted for malignant hematologic diseases. Inaddition, donor T cells provide protection from opportunisticinfections as evidenced by the high rate of infectious compli-cations in patients treated with T cell-depleted grafts or Tcell-depleting antibodies post transplantation. However, donorT cells are also responsible for the main complication afterallogeneic SCT, namely GVHD, which is still a major causeof treatment-related morbidity and mortality. In contrast toother improvements, progress concerning the prevention andtreatment of severe GVHD has been limited and it thereforestill represents the main obstacle for an extended use of allo-geneic SCT, e.g. for the treatment of serious autoimmune syn-dromes. Thus, separation of beneficial from harmful T celleffects is a major research goal in experimental and clinicalSCT.

The pathophysiology of GVHD is a multi-step processmainly driven by alloantigen specific T cells, but amplifiedby various contributions of the innate immune system as wellas treatment-related alterations in immune homeostasis [1].The pretreatment of the recipient with radio- and/or chemo-therapy, called “conditioning therapy”, is intended to createspace in the bone marrow compartment (myelosuppressionor myeloablation), to prevent host lymphocyte mediated graftrejection (immunosuppression) and to provide anti-tumoractivity in patients with malignant diseases (tumor cytotoxictherapy). Depending on the intensity of the pretreatment, thetherapy-induced tissue damage creates a pro-inflammatoryenvironment even before donor T cells are transfused. Thisenvironment is characterized by dysregulated cytokine secre-tion, altered chemokine and chemokine receptor expressionand upregulation of adhesion as well as MHC molecules inGVHD target tissues, all of which are factors that unspecifi-cally favor the activation and expansion of transfused donorT cells. In addition, lymphopenia induced by the preparatoryregimens to protect from graft rejection also promotes theactivation and expansion of transplanted T cells throughmechanisms related to homeostatic proliferation [2]. Theallospecific T cell response is initiated once host-reactivedonor T cells are activated by professional antigen-presentingcells (APC). Depending on the MHC-disparity between donorand recipient several distinct mechanisms of allogeneic T cellresponses can be differentiated that contribute to variousdegrees to the development of GVHD. In MHC-mismatchedSCT donor T cells recognize major histocompatibility anti-gens that consist of mismatched MHC/peptide complexes.Depending on the degree of disparity up to 10% of the donorT cell population can respond to these alloantigens with high

avidity and provoke a massive response against host tissues[3]. Such a response of donor T cells to mismatched host MHCis termed “direct allorecognition”. In MHC-matched SCT,so-called minor histocompatibility antigens (mHA) have thepotential to induce GVHD. mHA consist of MHC-presentedpeptides derived from polymorphic proteins that are differ-entially expressed in the donor and the recipient. Since donorT cells were not rendered tolerant towards these host-specificpeptides during their thymic development, they can respondto such antigens within the host. Thereby, mHA can be pre-sented by either residual host APC or by donor APC, oncehostAPC are replaced after hematopoietic reconstitution. Theresponse of donor T cells to host-specific peptides presentedby donor-derived (autologous) APC is referred to as “indirectallorecognition”. The severity of GVHD in mHA-mismatchedtransplantation models depends on the genetic disparitybetween donor and recipient and can range from minimaleffects to rapidly lethal GVHD that is indistinguishable fromthat seen after MHC-mismatched transplantation. Recently,Shlomchik et al. [4] have shown in a mHA-mismatched, CD8+

T cell-dependent model that residual host APC alone are suf-ficient to induce acute GVHD, while donor APC cross-presenting host antigens only contribute to a lower degreeonce GVHD is initiated [5]. In similarly designed experi-ments, Teshima et al. [6] demonstrated in an MHC classII-mismatched model that residual host APC are also in thissituation the main inducers of GVHD while MHC class IIexpression on GVHD target tissues is dispensable. In sum-mary, these studies revealed the essential role of residual hostAPC for the activation of allogeneic donor T cells leading totheir dramatic early expansion and differentiation into effec-tor cells. Once activated, such effector T cells most fre-quently affect skin, gut, liver and the lymphatic system, buttissue destruction can also occur in many other organs at vari-ous frequencies.

3. Protective role of CD4+CD25+ regulatory T cellsin GVHD

CD4+ T cells that constitutively express the a-chain of theIL-2 receptor (CD25) constitute about 5–10% of the CD4+ Tcell pool in peripheral blood and lymphoid organs.Apart fromtheir CD25 expression, CD4+CD25+ Treg cells are character-ized by the preferential but non-exclusive expression of vari-ous molecules, such as constitutive intracellular cytotoxic Tlymphocyte-associated antigen-4 (CTLA-4), glucocorticoid-induced TNF receptor family-related gene (GITR) andforkhead/winged-helix transcriptional regulator (Foxp3). Themain functional characteristics of CD4+CD25+ Treg cells aretheir reduced proliferative response to standard T cell stimu-lation (anergy) and their potent suppressive activity. Suppres-sion mainly refers to the capacity of Treg cells to inhibit theproliferation and cytokine secretion of conventional CD4+ andCD8+ T cells in a cell contact-dependent manner, althoughinhibitory effects on APC [7] and B cells [8] have also been

1067P. Hoffmann et al. / Microbes and Infection 7 (2005) 1066–1072

described. The molecular mechanism of suppression byCD4+CD25+ Treg cells has not yet been identified, but theinduction of their suppressive activity crucially depends ontheir own activation. The fact that CD4+CD25+ T cells withphenotypic and functional characteristics of Treg cells couldbe isolated from the thymus and co-evolve during thymic Tcell development led to their classification as “natural” Treg

cells, which separates them from “induced” Treg cells that arederived from conventional non-regulatory T cells in vitro orin vivo under variable treatment conditions and that mainlymediate their suppressive effects via the secretion of immu-nosuppressive cytokines [9]. In vivo, natural CD4+CD25+ Treg

cells restrict peripheral self-reactivity and protect from anexcessive expansion of self- as well as non-self-reactive Tcells in various disease settings [10].

The unique functional characteristics of naturalCD4+CD25+ Treg cells prompted several investigators to studytheir capacity to modulate alloresponses. In contrast to CD25–

T cells, CD4+CD25+ T cells did not proliferate after alloge-neic stimulation in vitro, but they dose-dependently sup-pressed the proliferation of conventional T cells in mixed lym-phocyte cultures. When examined in model systems of acuteGVHD, these observations held true in vivo, since purifieddonor-derived CD4+CD25+ T cells did not induce signs ofGVHD in mice even when transplanted in high numbers andacross complete MHC barriers. More importantly, the co-transplantation of donor CD4+CD25+ T cells together withan otherwise lethal dose of conventional (CD25–) donor Tcells at a 1:1 ratio ameliorated GVHD after MHC classII-mismatched or completely MHC-mismatched BMT[11–13]. The protective effect in the different model systemsranged from reduced loss of body weight and prolongationof mean survival time to complete inhibition of clinical GVHDresulting in the long-term disease-free survival of recipientanimals. Interestingly, protection from GVHD was achievedeven after delayed transfer of Treg cells, provided the courseof GVHD in the chosen model was not too aggressive [14]. Itthus seems that CD4+CD25+ Treg cells not only prevent analloresponse, but also modulate and potentially revert earlydisease stages, as recently also shown in a murine model ofinflammatory bowel disease [15]. Inhibition of GVHD wasonly observed when donor, but not host CD4+CD25+ Treg cellswere co-transplanted, and was achieved by either freshly iso-lated and unmanipulated [11] or by in vitro pre-activated andexpanded CD4+CD25+ Treg cells [12–14].

The most prominent effect observed in transplant recipi-ents that were protected from lethal GVHD by co-transplantedCD4+CD25+ Treg cells was an efficient inhibition of the earlyexpansion of alloreactive conventional CD4+ and CD8+ donorT cells [16,17]. Thus, restricting the clone size of alloaggres-sive T cells early after BMT seemed to be the main mecha-nism by which CD4+CD25+ Treg cells mediated protectionfrom lethal GVHD. Reduced numbers of donor-derived con-ventional T cells were not only recovered from lymphoidorgans (spleen and mesenteric lymph nodes), but also fromGVHD target tissues (liver and gut) of recipients when

CD4+CD25+ T cells were co-transplanted. Thus, inhibitionof T cell expansion by donor Treg cells could have either beenthe result of equal suppressive activity at all sites or of pre-dominant suppression in lymphoid organs followed by areduced export of alloaggressive T cells into GVHD targettissues. To address this question experimentally, we and oth-ers took advantage of the fact that CD62L expression is nec-essary for efficient lymph node entry of T cells. When theCD4+CD25+ Treg cell population was subdivided into aCD62L positive and a CD62L negative fraction and exam-ined in vitro, both subpopulations showed typical character-istics of Treg cells, namely equally high expression of foxp3,hyporesponsiveness to stimulation (anergy), lack of IL-2 secretion upon stimulation and equal suppressive activityafter polyclonal or allogeneic stimulation. In vivo, however,the CD62L+ subpopulation of CD4+CD25+ Treg cells sup-pressed the expansion of alloaggressive T cells after alloge-neic BMT much more efficiently and only the CD62L+ sub-population protected from lethal GVHD. In combination withthe finding that the CD62L+ subpopulation of Treg cells alsomigrated in much higher frequency to relevant secondary lym-phoid organs (e.g. mesenteric lymph nodes), these results sug-gested that regional lymph nodes are indeed the crucial sitesof interaction between alloaggressive and host-protectivedonor T cells for suppression of GVHD [17,18]. Since theseare also the sites of GVHD initiation, it is plausible thatresidual hostAPC there not only activate alloaggressive donorT cells, but also provide the relevant antigens, costimulatorymolecules and cytokine milieu to activate adoptively trans-ferred donor CD4+CD25+ Treg cells such that they gain theirsuppressive activity in close temporal and spatial proximityto their potential targets.

The ability of donor CD4+CD25+ Treg cells to restrict thealloantigen-driven T cell expansion required for the induc-tion of acute GVHD suggested that beneficial donor T celleffects, such as facilitation of stem cell engraftment and graft-versus-leukemia/lymphoma activity (GVL), might also beabrogated. However, it could be shown that neither residualhost CD4+CD25+ Treg cells nor donor CD4+CD25+ Treg cellstransplanted with the BM graft interfere with stem cell engraft-ment in various mouse models, but rather facilitate hemato-poietic reconstitution and the development of full donor chi-merism [19–21]. To study the influence of Treg cells on thecytotoxic activity of alloantigen-primed T cells, several invitro and in vivo studies were performed. Responder T cellsprimed in the presence or absence of Treg cells to MHC-mismatched MNC were tested for cytotoxic activity againststimulator-type lymphoma targets. Although expansion ofresponder T cells, and in particular CD8+ T cells, in responseto MHC-mismatched stimulator cells was inhibited whenCD4+CD25+ Treg cells were present in the co-culture, neithertheir initial activation nor their cytotoxic activity on a per cellbasis were diminished when compared to T cells primed inthe absence of Treg cells [22]. In the corresponding in vivomodels, lymphoma or leukemia bearing hosts were used astransplant recipients to evaluate the influence of Treg cells on

1068 P. Hoffmann et al. / Microbes and Infection 7 (2005) 1066–1072

the GVL-activity of co-transplanted conventional donor Tcells. As expected, donor Treg cells alone did not induce clini-cal GVHD when co-transplanted with T cell-depleted BM,but all animals died from tumor relapse due to the inability ofTreg cells to mediate GVL effects. In contrast, all tumor-bearing mice that were protected from lethal GVHD by theadministration of a 1:1 ratio of Treg cells and conventional Tcells eradicated their lymphoma, whereas those that receivedonly conventional donor T cells rapidly died from GVHD.These findings not only confirmed the results obtained in vitro,but also revealed that the excessive early expansion of allo-aggressive T cells (responsible for the progression to clinicalGVHD) was not required for donor T cell-mediated GVL-effects. As residual host hematopoietic cells are the preferen-tial and most sensitive targets of an alloresponse (graft-versus-hematopoiesis effect), eradication of residual lymphoma orleukemia can thus be achieved in the absence of clinicalGVHD [14,19,22]. This finding is supported by the clinicalexperience that conversion of mixed chimeras to full donorchimerism often occurs in the absence of GVHD after donorlymphocyte infusions, which are usually applied for the treat-ment of leukemia relapse in transplanted patients. In sum-mary, the animal models suggest that adoptively transferreddonor Treg cells interfere with the pathophysiology of acuteGVHD by restricting the progression of a subclinical allore-sponse to overt clinical GVHD without inducing completeparalysis of the T cell compartment. Thus, a fine-tuned bal-ance between alloaggressive and host-protective immunemechanisms determines the outcome of GVL-permissive tol-erance induction by donor CD4+CD25+ Treg cells in alloge-neic BMT.

4. The pool size model of peripheral toleranceinduction

Deduced from their studies in solid organ transplantation,Li et al. [23] previously described the specific problems ofpathogenic alloresponses and proposed the “pool size model”of peripheral tolerance induction. Summarized in brief theystate that in allogeneic organ transplantation a large pool ofresponding host T cells is readily available that promptly rec-ognizes intact allo-MHC or multiple mHA mismatches. Thesize of the responding T cell pool is further increased by thedramatic early expansion of alloantigen specific T cells thatdifferentiate into cytotoxic effector cells and usually causeacute graft rejection. In transplantation models that permitearly graft survival, a retraction of the alloaggressive T cellpool is regularly observed which is attributed to apoptoticmechanisms in responding T cells either by activation inducedcell death (AICD) or by passive cell death (PCD) [24]. Despitethese deletional mechanisms, the burden of alloaggressive Tcells regularly dominates suppressive immune mechanismsthat co-evolve during the course of an alloresponse [25]. Oneof those important suppressive mechanisms is the develop-ment of regulatory T cells. However, under “normal” trans-

plantation conditions they either evolve too late or are insuf-ficiently dominant to protect from organ damage, in thisscenario chronic graft rejection. The authors concluded fromthese observations that any intervention aimed at facilitatingperipheral tolerance to allogeneic grafts has to restrict the ini-tial expansion of these alloaggressive T cells to permit intrin-sic immunoregulatory mechanisms, in particular regulatoryT cells, to evolve then dominate a low, but not a high burdenof alloreactive T cells.

Applied to allogeneic BMT, this model is suited to illus-trate some of the findings concerning the role of adoptivelytransferred Treg cells in murine GVHD. Without the additionof donor CD4+CD25+ Treg cells, conventional donor T cellsco-transplanted with the stem cell graft encounter residualhost APC and dramatically expand within the first few daysafter transplantation (Fig. 1). As detailed above, this expan-sion of allospecific T cells is a hallmark in the induction phaseof acute GVHD (and even more pronounced in BMT as com-pared to solid organ transplantation) and a prerequisite forprogression to clinical or even lethal disease. As seen afterorgan transplantation, a contraction of the reactive T cell poolregularly occurs in mice that survive the early phase of acuteGVHD. This process, however, does not prevent further tar-get tissue destruction in gut, liver and skin, which then causesthe typical clinical symptoms and eventually the delayed deathfrom GVHD. The few Treg cells normally present in a stemcell graft are insufficient for protection from GVHD as thelarge pool of alloaggressive donor T cells always dominatesthem. Similarly, Treg cells generated de novo from the stemcell graft [26] evolve either too late or are insufficiently activeto protect from organ damage. Furthermore, GVHD itselfmight interfere with this de novo generation of Treg cells dueto disease involvement of lymphoid organs, in particular thethymus.

In contrast, co-transplantation of large numbers of donorCD4+CD25+ Treg cells at the time of BMT seems to influencethe pathophysiology of GVHD at several levels (Fig. 1): First,

Fig. 1. Shifting the balance towards immunoregulation early after transplan-tation by the co-transplantation of increased numbers of donor CD4+CD25+

Treg cells restricts the expansion of alloaggressive donor T cells and therebyfacilitates the development of stable immune tolerance. Adapted from Ref.[23], with kind permission from Elsevier.

1069P. Hoffmann et al. / Microbes and Infection 7 (2005) 1066–1072

the administered Treg cells, which probably recognize alloan-tigen with a similar precursor frequency as conventional Tcells [27,28], increase the initial pool size of suppressive cells.Second, the transplantation of increased numbers of Treg cellstogether with conventional T cells in the stem cell graft ensuresthat suppressive mechanisms are readily available at the timeand at the sites of GVHD induction thereby interfering withthe early phase of T cell priming. Most importantly, theco-transplanted Treg cells themselves restrict the expansionof the alloaggressive T cell pool, thereby preventing progres-sion of the alloresponse to apparent clinical or lethal GVHD.The suppression of donor T cell expansion without the needfor additional immunological or pharmacological interven-tions further ensures that Treg cell function is not abrogatedby such medication and that the IL-2 dependent deletionalmechanisms (AICD) in the alloaggressive T cells remain intact(in contrast to the situation during some of the currently usedimmunosuppressive regimens, such as treatment with cy-closporineA) [29].Although not yet shown in detail for BMT,additional effects of donor Treg cells described in other dis-ease models might also contribute to the protection fromGVHD. For example, the interaction of Treg cells with con-ventional T cells might not only limit their proliferative capac-ity, but also alter their functional program. Possible alter-ations might involve their cytokine production, theirrequirements for co-stimulation, the expression of adhesionmolecules and chemokine receptors affecting their migratorybehavior, or even their transformation into regulatory T cells(infectious tolerance) [30]. Similarly, it is likely that trans-ferred Treg cells not only act on co-transplanted conventionalT cells in a cell contact-dependent manner, but that theircontact-dependent as well as cytokine-mediated effects alsoaffect other cell populations, such as APCs, thereby modulat-ing the pro-inflammatory activity of those cells and theircapacity to present alloantigen.

In summary, the size and kinetics of immunosuppressivevs. alloaggressive immune reactions seem to be crucial fac-tors for the clinical outcome after allogeneic BMT and shift-ing the balance between these two competing components ofan immune response early after transplantation might facili-tate the development of stable tolerance.

5. Human CD4+CD25+ Treg cells and alloreactivity

Whether the findings from murine BMT models hold truefor human allogeneic SCT is unknown thus far, since compa-rable clinical trials have not yet been performed. However,human Treg cells can be found in peripheral blood, lymphoidorgans, cord blood and the thymus [31,32]. In contrast tomurine cells, human CD4+ T cells are comprised of three dif-ferent cell populations with respect to CD25 expression,namely CD25 negative, intermediate (int) and high express-ing cells. The Treg cell population seems to reside primarilywithin the CD25high subpopulation [33], while CD4+CD25int

cells contain many recently activated T cells. CD4+CD25high

T cells differ from CD4+CD25int T cells not only with regardto their CD25 expression, but also in their CD4 expressionlevel and FSc properties and can thus be separated from thelatter population under steady state conditions. A distinctionbetween CD25high and CD25int T cells, however, is not alwaysmade in studies on human Treg cells, which hampers the inter-pretation of respective data. Like murine CD4+CD25+ Treg

cells, human CD4+CD25high Treg cells are characterized by aconstitutive and high yet non-exclusive surface expression ofGITR and intracellular expression of CTLA-4. Foxp3, thusfar the most specific marker for murine Treg cells and shownto be required for their development [34–36], is likewise pref-erentially expressed by human CD4+CD25high T cells,although it can also be detected at low levels in CD25– Tcells upon stimulation [37]. Whether those cells representfreshly induced Treg cells [38] or whether their foxp3-expression occurs independent of any suppressive activity hasto await further clarification. Human CD4+CD25high T cellsalso share functional characteristics with murine CD4+CD25+

Treg cells, i.e. reduced proliferation after standard T cell stimu-lation (anergy), diminished capacity for cytokine secretionand profound suppressive activity after activation. Compa-rable to murine Treg cells, they suppress the proliferativeresponse of CD25– T cells in vitro in mixed lymphocyte reac-tions and their suppressive activity depends on cell contactbut not on secreted cytokines [28,32,39]. As for murine Treg

cells, the molecular basis of suppression of humanCD4+CD25high T cells has still not been clarified in detail,but involvement of membrane-bound TGF-b as well asCTLA-4 has been reported [32]. Overall, these findings indi-cate that CD4+CD25high T cells in fact represent the humancounterpart of murine CD4+CD25+ Treg cells and that theymight have a similar relevance for the modulation of immuneresponses in humans as murine Treg cells have in variousexperimental disease models.

A series of recent studies suggested that altered numbersor function of CD4+CD25high Treg cells are associated withcertain diseases in humans. This was demonstrated most con-vincingly in patients with mutations in the foxp3 gene whosuffer from a multi-organ autoimmune syndrome (IPEX:immune dysregulation, polyendocrinopathy, enteropathy,x-linked syndrome) [10]. Similarly, a few studies publishedrecently tried to explore the role of CD4+CD25high Treg cellscontained in low numbers in stem cell grafts by monitoringand comparing their reconstitution after SCT in patients withand without acute or chronic GVHD [40–42]. As expected,results and conclusions varied and probably reflect differ-ences in study design, patient selection, conditioning regi-mens, underlying diseases, co-medication, especially immu-nosuppressants and, most importantly, the difficulty to reliablyidentify and quantify Treg cells in patients with ongoingGVHD or treatment-related changes in the lymphoid com-partment during immune reconstitution. Thus, the lack ofexclusive surface markers for Treg cells represents a majorhurdle for such studies, especially since phenotypic Treg cellcriteria validated with cells from healthy volunteers are not

1070 P. Hoffmann et al. / Microbes and Infection 7 (2005) 1066–1072

always considered and might not even be applicable forpatient-derived samples or previously frozen cell isolates. Tocircumvent this problem, Miura et al. [40] measuredfoxp3 mRNA levels in PBMC and found an inverse correla-tion with acute GVHD development in patients undergoingBMT. Using phenotypic Treg criteria (high expression levelsof CD25), a similar trend was reported by Sanchez et al. inacute GVHD, while both, Sanchez et al. and Clark et al.detected increased numbers of CD4+CD25+ Treg cells inpatients suffering from chronic GVHD as compared to thosenot developing chronic GVHD or to normal volunteers[41,42]. The decision which of these findings merely repre-sent epiphenomena and which are direct cause or conse-quence of the activity of transplanted Treg cells has to awaitfurther investigations. Since the few cells contained in a con-ventional stem cell graft obviously do not prevent disease [43],carefully designed clinical trials have to test the concept ofwhether the co-transplantation of large numbers of donorCD4+CD25high T cells early after SCT promotes peripheraltolerance induction. Such trials are currently in preparationat several institutions. The recently described methods forpolyclonal or alloantigen specific in vitro expansion of humanTreg cells will greatly facilitate the generation of appropriatecell numbers for such studies [28,44,45].

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